The insulin-like growth factor II (IGFII) is a positive regulator of embryonic growth. It is suspected that higher levels of IGFII are responsible for the growth abnormalities and tumor predisposition associated with a human genetic disorder, the Beckwith-Wiedemann syndrome. The potent mitogenic effects of IGFII, its expression in normal and malignant breast tissues and its effects on breast cancer lines grown in vitro or as xenografts in nude mice have led to the suggestion that IGFII is a mediator of the mitogenic effects of estrogen, and that autocrine stimulation by IGFII may play a role in the progression of these tumors to an estrogen-independent growth phase. However, in vitro experimental systems cannot reproduce the complexities of growth regulation in vivo, where the stimulatory effects of IGFII can be attenuated, or altogether abolished, e.g. by factors that influence the availability of IGFII for binding to its receptor. To analyze the effects of an increased level of IGFII in vivo, we will create transgenic lines that carry extra copies of the insulin-like growth factor-2 gene (Igf2). These mice will represent an animal model for mutations that result in increased production of IGFII. Similar effects, by several different mechanisms, are believed to be achieved in the BWS in humans. To create IGFII overproducing mice we will first define the regulatory elements that are required for proper expression of the transgenes. The search for regulatory regions of Igf2 will be carried out by linking different segments of the Igf2 gene and its flanks to a reporter gene (the bacterial beta-galactosidase gene), injecting the Igf2lacZ clones into mouse embryos, and analysis of the transgenic offspring for lacZ expression. Comparison of the genomic regions present in clones that are properly expressed, and clones that are not, will allow us to define the position and span of the regulatory elements. To create Igf2 overexpressing lines, we will introduce Igf2 clones that retain the coding region of the gene, linked to the regulatory elements required for proper in vivo expression, into mouse embryos. Several strategies will be used to overcome the toxic effects of Igf2 on the embryos: 1) reversible suppression of transgene activity by n vitro methylation of the injected DNA; 2) injection of Igf2 clones into embryonic stem cells, and injection of the cells into blastocysts to create chimeras between normal and IGFII overproducing cells; 3) placing the gene under the control of a tissue-specific promoter that will result in later activation and mosaic expression of the transgene. To target Igf2 expression to the mammary glands, we will use a mouse mammary tumor long terminal repeat enhance. Our phenotypic analysis of the transgenic progeny will focus on growth abnormalities (e.g. differences in overall body size, or size of Igf2 expressing organs), and on the frequency and type of tumors that develop in the transgenic offspring. The results of these studies will contribute to our understanding of the roe played by IGFII in tumorigenesis, and its suitability as a potential target for antitumor therapies.